Cocaine use during pregnancy has been associated with numerous adverse perinatal outcomes. Among other effects, cocaine clearly predisposes the fetus and neonate to various cardiovascular dysfunctions. Our preliminary studies indicated that prenatal cocaine exposure caused an increase in apoptosis in near-term fetal rat heart and a decrease in cardiac contractility in newborn rats. Compelling evidence indicates that apoptosis plays a key role in heart development and in several cardiovascular diseases. Yet the cellular/molecular mechanisms underlying cocaine-induced apoptosis in the developing heart are unknown. The proposed studies focus on these mechanisms, and will address the general hypothesis that cocaine increases apoptosis in myocardial cells of the developing heart through nitric oxide (NO) and mitogen-activated protein kinases (MAPKs), leading to mitochondrial cytochrome c release and subsequent activation of the caspase cascade. Four of its main corollaries will be addressed by 4 Specific Aims which will test whether cocaine 1) activates constitutive nitric oxide synthase (NOS) and up-regulates inducible NOS (iNOS), resulting in apoptosis, 2) increases the balance of activities of p38 MAPK/JNK versus ERK resulting in apoptosis, 3) affects Bcl-2 family proteins by increasing the balance of proapoptotic/antiapoptotic proteins and inducing the translocation of proapoptotic proteins to mitochondria, and 4) induces mitochondrial cytochrome c release and subsequent activation of the caspase cascade. To achieve these aims, we propose a series of experiments in primary cultures of fetal rat cardiac myocytes. We will measure NO release and expression of eNOS, nNOS, and iNOS; activities of p38 MAPK, JNK, and ERK; protein levels and subcellular distribution of Bcl-2, Bcl-xL, Bax, and Bad; mitochondrial cytochrome c release; and activities of caspase-3, caspase-8, and caspase-9. The results of the proposed studies will provide a comprehensive and novel assessment of the dynamic interactions among nitric oxide, MAPKs, Bcl-2 family proteins, mitochondrial cytochrome c, and the caspase cascade in cocaine-induced myocyte apoptosis, and will improve our understanding of the adverse effects of cocaine on the developing heart. They will also provide exciting new information to fill the important gaps in our understanding of signaling mechanisms in myocyte apoptosis in general. Such an understanding has obvious clinical implications because the increasing information has pointed to an important role of apoptosis in cardiovascular diseases.